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Abstract

Confocal Raman microscopy as a label-free technique was applied to study the uptake
and internalization of poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) and carbon nanotubes (CNTs) into hepatocarcinoma
human HepG2 cells. Spontaneous confocal Raman spectra was recorded from the cells
exposed to oxidized CNTs and to PLGA NPs. The Raman spectra showed bands arising from
the cellular environment: lipids, proteins, nucleic acids, as well as bands characteristic
for either PLGA NPs or CNTs. The simultaneous generation of Raman bands from the cell
and nanomaterials from the same spot proves internalization, and also indicates the
cellular region, where the nanomaterial is located. For PLGA NPs, it was found that
they preferentially co-localized with lipid bodies, while the oxidized CNTs are located
in the cytoplasm.

Introduction

The use of nanomaterials and nanoparticles (NPs) in medicine as drug delivery vectors,
sensors or contrast agents is among the most promising areas in nanotechnology research.
For the application of nanotechnology in medicine, 'in vitro' work is of paramount importance, especially regarding the assessment of possible
toxicological consequences of the nanomaterials/NPs. It is a key issue to study the
effects of 'nano' in the cellular machinery and to understand how the nanomaterials
are processed in the cell, and their distribution and fate, after being taken up by
the cells. Confocal laser scanning microscopy (CLSM) is often applied for uptake studies,
but its application for NPs is not always easy, since the size of the NPs falls well
below optical resolution. Also, a main drawback of CLSM is that, for most of the cases,
both the NPs and cellular compartements must be fluorescently labelled, and this is
not always an easy task. Besides that, labelling of NPs may in some cases require
complex chemical routes including, for example, silanization, assembly of polymers,
etc. As a result, the labelling can induce significant changes in the structure and
properties of NPs, which may affect uptake and toxicity. Transmission electron microscopy
(TEM) can be used to study the uptake and localization of NPs and nanomaterials, avoiding
their labelling. The drawback of TEM for this application is that it requires complex
and time-demanding preparations that also may affect the localization of the nanomaterials
within the cell. Other label-free techniques for the study of the localization of
nanostructures within cells are spontaneous Raman microscopy and coherent anti-stokes
Raman (CARS) microscopy. In CARS, a single Raman band coming from the nanomaterial
is scanned throughout the cell. A mapping of the cell is obtained showing the intensity
distribution of the chosen Raman band [1]. Drawbacks of CARS are that only selected bands can be mapped, and furthermore, spectral
overlapping may cause problems.

Confocal Raman microscopy (CRM) combines spontaneous Raman emission with confocal
detection. We will show here that CRM can be used to study the localization of nanomaterials
in the cells, taking advantage of the fact that in every spot the whole Raman spectrum
is recorded. The latter thus contains bands coming from the nanomaterials and from
representative cell molecules: proteins, DNA and lipids, which allow to identify the
region of the cell [2,3], where the nanostructures are located. This article is among the first [4] to explore the use of the spontaneous Raman emission for the detection of nanomaterials
inside cells and to assess the intracellular region from the spectra, where the nanomaterial
is located. Previous study with CRM and cells has focused in the recognition of different
cellular environment through their chemical fingerprints and the evaluation of changes
in metabolism [5,6]. Poly(lactide-co-glycolide) (PLGA) NPs and carbon nanotubes (CNTs) have been chosen as two representative
and remarkably different systems that can be studied with CRM. To our knowledge, this
is the first article where CRM is used for CNT detection at cellular level.

PLGA NPs were prepared by the O/W emulsion-solvent evaporation method [4]. Size and shape of the PLGA NPs were characterized by TEM (JEOL JEM 2100F, Japan)
[7]. Multiwalled CNTs were purchased from Proforma (USA). Oxidation of CNTs was achieved
as described in the literature by Zhang et al. [8].

Raman microscopy

Micro-Raman analyses were performed using a Renishaw inVia Raman Microscope. Measurements
were performed using a 532-nm laser excitation wavelength with a grating of 1800 mm-1. Most measurements were conducted using a ×40 water immersion objective with a focal
spot of approximately 1 μm in diameter. Spectra were recorded in the region 300-3600
cm-1 , with a resolution of approximately 7 cm-1. The system was calibrated to the spectral line of crystalline silicon at 520.7 cm-1. At least 8-15 accumulation scans, at different spots in the various cell compartments,
lipid bodies (LB), cytoplasm and nucleus, were used to reduce the spectral noise.
All spectra had a correction for the PBS solution and glass cover slip baseline. After
CNTs or NPs exposure and repeated washings with PBS, the Raman spectra were taken
only from cells, where no visible CNT aggregates could be observed.

Results

In Figure 1a, a characteristic TEM image of the PLGA NPs is shown--the size of the NPs ranges
from approximately 250-400 nm. PLGA NPs were labelled with Rd6G for visualization
in HepG2 cells with CLSM. In Figure 1b, the confocal images show that PLGA NPs are associated with the cells. This follows
from the red colour indicating the Rd6G-labelled NPs, distributed around the blue-stained
nucleus. The single confocal image does not unambiguously prove the internalization
of the PLGA NPs in the cells, which could also be associated to the cell membrane.
However, a scan in the z-direction (z-scan) could show the intracellular presence of the NPs, especially, if the plasma
membrane had been also stained [9].

Figure 1.Characterization and confocal fluorescence imaging of PLGA NPs in cells. (a) TEM morphology of PLGA NPs stabilized with PEI and (b)CLSM images of HepG2 cells
after being co-cultured with PLGA NPsstabilized with PEI.

CRM experiments were performed in the same experimental conditions, but without labelling
of the NPs, and are shown in Figure 2. In Figure 2a, representative Raman spectra, taken at different regions of the cell are shown.
The nucleus, cytoplasm and LB can be identified by their chemical signature provided
by the Raman spectrum. The symmetric stretch bands of CH2 (2850 cm-1) to CH3 (2935 cm-1) is much more intense in LB (blue line) than in the cytoplasm (green line) due to
a lower density of CH2 groups in proteins compared with lipids. The nucleus region was identified by the
smallest intensity ratio of CH2 to CH3 bands, as well as by bands assigned as vibration of DNA bases of adenine (A) and guanine
(G) (red line). The spectra in Figure 2b correspond to Raman spectra taken from HepG2 cell, before and after incubation with
PLGA NPs. A spectrum of the PLGA NPs taken in the dry state is also shown (pink curve).
It can be seen that the Raman spectrum of the cells, after incubation with PLGA, represents
a superposition of the PLGA particle spectrum (pink) and the spectrum of the control
(green). Besides the bands typical from lipids at 2850 and 2900 cm-1, which can be attributed to the LB, the intense CH2 and CH3 vibrations of PLGA are clearly visible. Looking at different spots in the cell, at
the same plane and at different positions regarding the z-direction, revealed that when bands characteristic for PLGA NPs were observed, the
typical LB signature was also present.

Figure 2.Confocal Raman imaging of HepG2 cells before and after exposure to PLGA NPs. (a) Raman spectrum recorded at different positions within a cell from the HepG2
line (ν indicates stretching and δ deformation vibration modes; l denote vibrations
of lipids and p of protein). (b) Spot Raman spectra (dark blue) in cells exposed to
PLGA NPs covered with PEI, pink and green lines denote the component spectra of PLGA
NPs and of the cells. The insets correspond to the image of the cell under study

Similar uptake experiments were performed with oxidized CNTs. The CNTs were oxidized
to provide them with charges to ensure their stabilization in aqueous solution. In
Figure 3a, we can observe the Raman spectrum of oxidized CNTs and HepG2 cells exposed to the
CNTs. CNTs show characteristic bands at 1350 cm-1 (D-band) and 1585 cm-1 (G-band) [10,11]. The D-band is an indicator for disorder in the graphene sheet and is called ''disorder-induced"
band. The G-band is a tangential mode originating from tangential oscillations of
the carbon atoms in the CNTs. These bands can be clearly observed in the cellular
spectra. Scans were performed at different planes within the cells as shown in Figure
3b. The plane denoted by 0 μm corresponds to the situation, where the signals of the
D and G bands from the CNTs were the strongest. Then, spectra were recorded at higher
and lower planes, respectively. In all cases, the CNTs spectral signature was parallelled
by CH3-stretching modes, typical for proteins. The CH2 stretching, which is indicative for LB, can be barely seen. The intensity of the CNTs
bands varied considerably over the different scan planes. From these findings, we
draw the conclusion that the CNTs are not homogeneously distributed in the cytoplasm,
nor they are closely associated with LB. Furthermore, the z-scanning provides an unambiguous proof of internalization of the NPs, since we move
in distances of micrometers inside the cell, where the detection of CNTs attached
to the cell membrane from the outside is very unlikely.

Figure 3.Raman spectra of CNTs and Raman imaging of cells exposed to CNTs. (a) Spectra of oxidized CNTs (green), free cell in the region of the LB (pink) and
cell exposed to CNTs at the same region (blue). (b) Raman spectra taken in one spot
at different planes at a HepG2 cell treated with oxidized CNTs. The insets correspond
to the image of the cell under study.

Conclusions

Spontaneous CRM has been successfully applied to identify PLGA NPs and oxidized CNTs
in single hepatocarcinoma cells, which had been co-cultivated with the NPs and CNTs.
The data prove that CRM, being a label-free technique, is a valuable tool to study
the uptake of nanomaterials into cells. For PLGA NPs, CRM confirms the observations
from CLSM and proves internalization. The z-scanning of the cells with CNTs reveals that these are incorporated in the cytoplasm
and are not co-associated with the LB.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

GR performed the synthesis of the PLGA nanoparticles and the surface modification
of the CNTs. She also conducted the Confocal Raman experiments. ER conducted the Confocal
Fluorescence Microscopy experiments and did the cell culture work. IE supported GR
with the Raman experiments and helped with the interpretation of the data. ED provided
support in the design of the experiments and the interpretation of the Raman spectra.
SEM conceived, designed and coordinated the study. All authors read and approved the
final manuscript.

Acknowledgements

This study was supported by the European Commission in the framework of FP7 Theme
4-NMP, Proposal No: CP-FP 228825-2 HINAMOX, as well as by the grant MAT2010-18995
from the Spanish Ministry of Science and Innovation. S.E. Moya is a Ramon y Cajal
Fellow.